2.16. Immunohistochemistry
For the immunohistochemistry of RIP3, the tissue were paraffin-coated
and sectioned with a paraffin microtome at a thickness of 5 μm.
Subsequently, the sections were dewaxed in xylene and hydrated in a
gradient ethanol solution. After dewaxing, antigen recovery was
performed in 10-mM sodium citrate buffer (pH 6.0) at 121°C for 15 min,
followed by 3% methanolic hydrogen peroxide solution for 15 min to
quench endogenous peroxidase activity. Then, the
slides were treated with 0.3%
Triton-X 100 to break the membrane for 15 min, and then washed with PBS
for three times. The slides were incubated with primary antibody at 4 °C
overnight. The next day, the slides were rewarmed for 10 min at RT and
then incubated with secondary antibody at RT for 2 h and exposed to DAB
chromogenic for 2-3 min. Finally, hematoxylin was used for staining cell
nuclei for 5 min. At last, the final product was visualized using a
microscopic imaging system.